Rapid and Sensitive Method of Forensic Toxicology in Post-Mortem Subjects and in Live and Post-Mortem Animals Using Oral Fluid Testing

ABSTRACT

The present invention provides a rapid, sensitive method for forensic drug testing in a post-mortem subject or a live or a post-mortem animal using oral fluid collected from the post-mortem subject or live or post-mortem animal. The method comprises collecting a sample of oral fluid from a post-mortem subject or a live or a post-mortem animal, analyzing the oral fluid sample qualitatively to detect the presence of one or more non-naturally occurring drugs, analyzing the oral fluid sample quantitatively to determine concentration of the one or more non-naturally occurring drugs in the post-mortem subject or in the live or the post-mortem animal, and identifying the one or more non-naturally occurring drugs in the post-mortem subject or in the live or the post-mortem animal. The detection and quantification in oral fluid is more sensitive and faster than detection and quantification of the non-naturally occurring drugs in blood, urine, bile, and liver tissue collected from the same post-mortem subject. Further, the qualitative and quantitative results are obtained in as little as three hours.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.15/785,703, filed Oct. 17, 2017, which is a continuation-in-part ofapplication Ser. No. 15/164,402, filed May 25, 2016, now U.S. Pat. No.9,817,006, which is a continuation-in-part of application Ser. No.14/744,324, filed Jun. 19, 2015, now U.S. Pat. No. 9,366,685, all ofwhich are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates to forensic toxicology in post-mortemsubjects. More particularly, the present invention relates to a rapidand sensitive method of drug testing in post-mortem subjects and liveand post-mortem animals using oral fluid collected from the subjects oranimals to quickly and sensitively detect the presence of, and quantifythe concentrations of, one or more drugs in the subjects or animals.

BACKGROUND OF THE INVENTION

In recent years, overdose from both licit and illicit drugs has been anincreasingly common cause of death in persons fifteen to seventy yearsof age. After a suspected drug death, a major objective at autopsy is todetermine whether any drugs measured in the decedent have played a rolein the cause of death. Currently, post-mortem forensic drug analysesrely upon traditional biological matrices such as blood, urine, bile,and liver tissue. The specific matrix used for specimen retrievaldepends, in part, on the time after death that the sample is collectedand the consistency of collection, which may vary due to differences inclotting time, fluid movement and changes in cellular components. Oncedeath has taken place, many drugs are released from their binding sitesin tissue as pH decreases and the process of autolysis proceeds. By thetime a sample has reached the clinical chemistry laboratory foranalysis, it may be unsuitable for analysis. For example, drugconcentrations in blood taken from an individual at one site may betwice the concentration as that taken at the same time from a differentsite (e.g., sublingual region versus femoral vein). In addition, indecaying cadavers, viable sample matrices typically are hard to retrieveand oftentimes are limited solely to putrefactive fluid in pleuralcavities and blisters. Sample collection from blood, urine and bodytissues also requires the use of protective gear to prevent possiblespread of infection. Further, sample analyses typically aretime-consuming, as various instrumentalities usually are employed forboth qualitative and quantitative analyses of the samples.

Further, methods to detect and quantify non-naturally occurring drugs inlive and post-mortem animals have long been needed. The number ofanimals that inadvertently ingest, or intentionally have been given,licit or illicit drugs has risen through the years. Currently, urinetesting has been successful to detect barbiturates, opiates,benzodiazepines, and amphetamines/methamphetamines in dogs. However,urine testing is not able to quantify the concentration of these andother non-naturally occurring drugs in animals.

There exists a need, therefore, for a fast, sensitive, and less invasivemethod to conduct forensic toxicology in post-mortem subjects, as wellas in live and post-mortem animals, than what is currently available.

SUMMARY OF THE INVENTION

The present invention fulfills this need by providing a rapid,sensitive, and less invasive method of forensic drug testing to detectand quantify non-naturally occurring drugs in post-mortem subjects, aswell as in live and post-mortem animals, using oral fluid collected frompost-mortem subjects and from live and post-mortem animals.

In an aspect of the invention, the method comprises collecting a sampleof oral fluid from a post-mortem subject, analyzing the oral fluidsample qualitatively to detect the presence of one or more non-naturallyoccurring drugs, analyzing the oral fluid sample quantitatively todetermine concentration of the one or more non-naturally occurring drugsin the post-mortem subject, and identifying the one or morenon-naturally occurring drugs in the post-mortem subject, whereindetection and quantification in oral fluid is more sensitive and fasterthan detection and quantification of the non-naturally occurring drugsin blood, urine, bile, and liver tissue collected from the samepost-mortem subject using the same qualitative and quantitative methods,and wherein qualitative and quantitative results are obtained in aslittle as three hours.

In another aspect of the invention, the method comprises collecting asample of oral fluid from a live or post-mortem animal, analyzing theoral fluid sample to detect the presence of and quantify theconcentration of the one or more non-naturally occurring drugs in thelive or post-mortem animal, and identifying the one or morenon-naturally occurring drugs in the live or post-mortem animal, whereindetection and quantification in oral fluid is more sensitive and fasterthan detection and quantification of the non-naturally occurring drugsin urine and blood, collected from the same live or post-mortem animalusing the same quantitative methods, and wherein detection andquantification are obtained in as little as three hours. In anembodiment of the invention, the animal is a dog, cat, horse, or anyother farm animal. In another embodiment of the invention, the animal isa dog.

The one or more non-naturally occurring drugs and drug metabolites thatmay be detected and quantified in accordance with the present inventionincludes, without limitation, drugs included in the following drugclasses: non-steroidal anti-inflammatory drugs (NSAIDs) including,without limitation, acetaminophen and aspirin; alcohol; alcoholmetabolites including, without limitation, ethyl glucuronide (EtG) andethyl sulfate (EtS); barbiturates including, without limitation,amobarbital, butabarbital, butalbital, pentobarbital, phenobarbital andsecobarbital; benzodiazepines including, without limitation, alprazolam,alpha-hydroxyalprazolam, oxazepam, 7-aminoclonazepam, diazepam,nordiazepam, midazolam, triazolam, temazepam, lorazepam and clonazepam;synthetic cannabinoids including, without limitation, “K2” or “spice;”cathinones including, without limitation, methylenedioxypyrovalerone(MDPV), methylone or mephedrone and Mitragyna speciose; generalanesthetics including, without limitation, ketamine and norketamine;muscle relaxants including, without limitation, carisoprodol,cyclobenzaprine and meprobamate; neuroleptics including, withoutlimitation, gabapentin and pregabalin; opiates including, withoutlimitation, codeine, hydrocodone, hydromorphone, morphine, oxycodone,pentazocine and oxymorphone; semi-synthetic opioids including, withoutlimitation, buprenorphine, fentanyl, meperidine, methadone,propoxyphene, o-desmethyl-cis-tramadol, tramadol and naltrexone; opioidantagonists/analgesics including, without limitation, naloxone andtapentadol; stimulants including, without limitation, amphetamine andmethylphenidate; hypnotics including, without limitation, zopiclone,zolpidem and zaleplon; antitussives including, without limitation,dextromethorphan; antidepressants including, without limitation,nortriptyline and amitriptyline; cannabinoids including, withoutlimitation, delta-9-tetrahydrocannabinol (THC); antipsychoticsincluding, without limitation, quetiapine; anticonvulsants including,without limitation, phenytoin and lamotrigine; antihistamines including,without limitation, diphenylhydramine; and illicit drugs including,without limitation, cocaine/benzoylecgonine, heroin/6-acetylmorphine,3,4-methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine(MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), methamphetamine andphencyclidine (PCP).

The sample of oral fluid is collected from either the buccal (i.e.,oral) cavity of the post-mortem subject or the live or post-mortemanimal, such as from the sublingual region (i.e., under the tongue), orfrom the submandibular region, such as the submandibular gland.Approximately one milliliter (mL) of oral fluid easily is collected inabout one minute to about ten minutes using a collection pad.

After collection of the oral fluid sample, it is analyzed qualitativelyusing an enzyme-linked immunosorbent assay (ELISA); and is analyzedquantitatively using liquid chromatography-mass spectrometry/massspectrometry (LC-MS/MS).

BRIEF DESCRIPTION OF THE DRAWINGS

A fuller understanding of the invention can be gained from the followingdescription when read in conjunction with the accompanying drawings, inwhich:

FIG. 1 is a bar graph showing concentrations of 6-acetylmorphine,alprazolam, codeine, methadone, and morphine collected from oral fluidof a post-mortem subject (PM-1) in accordance with an embodiment of theinvention, compared to concentrations of the same drugs detected inblood from this subject reported by Lab X;

FIG. 2 is a bar graph showing concentrations of 6-acetylmorphine,alprazolam, carisoprodol, fentanyl, morphine, and oxycodone collectedfrom oral fluid of a post-mortem subject (PM-2) in accordance with anembodiment of the invention, compared to concentrations of the samedrugs detected in blood from this subject reported by Lab X;

FIG. 3 is a bar graph showing concentrations of 6-acetylmorphine,benzoylecgonine, carisoprodol, codeine, fentanyl, and morphine collectedfrom oral fluid of a post-mortem subject (PM-3) in accordance with anembodiment of the invention, compared to concentrations of the samedrugs detected in blood from this subject reported by Lab X;

FIG. 4 is a bar graph showing concentrations of 6-acetylmorphine,codeine, and morphine collected from oral fluid of a post-mortem subject(PM-4) in accordance with an embodiment of the invention, compared toconcentrations of the same drugs detected in blood from this subjectreported by Lab X;

FIG. 5 is a bar graph showing concentrations of clonazepam and fentanylcollected from oral fluid of a post-mortem subject (PM-5) in accordancewith an embodiment of the invention, compared to concentrations of thesame drugs detected in blood from this subject reported by Lab X;

FIG. 6 is a bar graph showing concentrations of 6-acetylmorphine,fentanyl, and morphine collected from oral fluid of a post-mortemsubject (PM-6) in accordance with an embodiment of the invention,compared to concentrations of the same drugs detected in blood from thissubject reported by Lab X;

FIG. 7 is a bar graph showing the concentration of fentanyl collectedfrom oral fluid of a post-mortem subject (PM-7) in accordance with anembodiment of the invention compared to the concentration of the samedrug detected in blood from this subject reported by Lab X;

FIG. 8 is a bar graph showing concentrations of 6-acetylmorphine,codeine, cyclobenzaprine, morphine, and oxycodone collected from oralfluid of a post-mortem subject (PM-8) in accordance with an embodimentof the invention, compared to concentrations of the same drugs detectedin blood from this subject reported by Lab X;

FIG. 9 is a bar graph showing concentrations of methadone, morphine andquetiapine collected from oral fluid of a post-mortem subject (PM-9) inaccordance with an embodiment of the invention, compared toconcentrations of the same drugs detected in blood from this subjectreported by Lab X;

FIG. 10 is a bar graph showing concentrations of cyclobenzaprine,hydrocodone, methadone, o-desmethyl-cis-tramadol, oxycodone, andoxymorphone collected from oral fluid of a post-mortem subject (PM-10)in accordance with an embodiment of the invention, compared toconcentrations of the same drugs detected in blood from this subjectreported by Lab X;

FIG. 11 is a bar graph showing concentrations of 6-acetylmorphine,benzoylecgonine, buprenorphine, fentanyl, and morphine collected fromoral fluid of a post-mortem subject (PM-11) in accordance with anembodiment of the invention, compared to concentrations of the samedrugs detected in blood from this subject reported by Lab X;

FIG. 12 is a bar graph showing concentrations of 6-acetylmorphine,benzoylecgonine, codeine, fentanyl, hydrocodone, and morphine collectedfrom oral fluid of a post-mortem subject (PM-12) in accordance with anembodiment of the invention, compared to concentrations of the samedrugs detected in blood from this subject reported by Lab X;

FIG. 13 is a bar graph showing the concentration of hydrocodonecollected from oral fluid of a post-mortem subject (PM-13) in accordancewith an embodiment of the invention, compared to the concentration ofthe same drug detected in blood from this subject reported by Lab X;

FIG. 14 is a bar graph showing concentrations of alprazolam,benzoylecgonine, and fentanyl collected from oral fluid of a post-mortemsubject (PM-14) in accordance with an embodiment of the invention,compared to concentrations of the same drugs detected in blood from thissubject reported by Lab X;

FIG. 15 is a bar graph showing concentrations of 6-acetylmorphine,fentanyl, and morphine collected from oral fluid of a post-mortemsubject (PM-15) in accordance with an embodiment of the invention,compared to concentrations of the same drugs detected in blood from thissubject reported by Lab X;

FIG. 16 is a bar graph showing the concentration of gabapentin collectedfrom oral fluid of a post-mortem subject (PM-16) in accordance with anembodiment of the invention, compared to concentrations of the same drugdetected in blood from this subject reported by Lab X;

FIG. 17 is a bar graph showing the concentration of naloxone collectedfrom oral fluid of a post-mortem subject (PM-17) in accordance with anembodiment of the invention, compared to the concentration of the samedrug detected in blood from this subject reported by Lab X;

FIG. 18 is a bar graph showing the concentration of tramadol collectedfrom oral fluid of a post-mortem subject (PM-18) in accordance with anembodiment of the invention, compared to the concentration of the samedrug detected in blood from this subject reported by Lab X;

FIG. 19 is a bar graph showing the concentration of acetaminophencollected from oral fluid of a post-mortem subject (PM-19) in accordancewith an embodiment of the invention, compared to the concentration ofthe same drug detected in blood from this subject reported by Lab X;

FIG. 20 is a bar graph showing concentrations of the alcohols ethylsulfate (EtS) and ethyl glucuronide (EtG) collected from oral fluid of apost-mortem subject (PM-20) in accordance with an embodiment of theinvention, compared to concentrations of the same drugs detected inblood from this subject reported by Lab X;

FIG. 21 is a bar graph showing the concentration of ketamine collectedfrom oral fluid of a live dog (SF10085) in accordance with an embodimentof the invention;

FIG. 22 is a bar graph showing the concentration of ketamine collectedfrom oral fluid of a live dog (SF10086) in accordance with an embodimentof the invention;

FIG. 23 is a bar graph showing the concentration of ketamine collectedfrom oral fluid of a live dog (SF10087) in accordance with an embodimentof the invention;

FIG. 24 is a bar graph showing the concentration of ketamine collectedfrom oral fluid of a live dog (SF10088) in accordance with an embodimentof the invention;

FIG. 25 is a bar graph showing the concentration of ketamine collectedfrom oral fluid of a live dog (SF10092) in accordance with an embodimentof the invention;

FIG. 26 is a bar graph showing the concentration of ketamine collectedfrom oral fluid of a post-mortem dog (SF10093) in accordance with anembodiment of the invention;

FIG. 27 is a bar graph showing the concentration of methamphetaminecollected from oral fluid of a live dog (SF10048) in accordance with anembodiment of the invention; and

FIG. 28 is a bar graph showing the concentrations of diazepam, ketamine,acetaminophen, tramadol and gabapentin collected from oral fluid of apost-mortem dog (SF10049) in accordance with an embodiment of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The following investigation was a prospective and controlled study oftwenty autopsy cases from post-mortem subjects designed in order todocument the efficacy, accuracy, and rapidity of using oral fluid todetect the presence of (i.e., screen) and quantify drug concentrationsin forensic autopsies compared to conventional collection modalitiesused in forensic autopsies, which use blood, urine, bile, and livertissue. In addition, an investigation was undertaken to document theefficacy, accuracy and rapidity of using oral fluid to quantify drugconcentrations in six live dogs and in two post-mortem dogs.

As used herein, the term “post-mortem subject” is defined as a humanbeing.

As used herein, the term “analytes” refers to all non-naturallyoccurring drugs except alcohol and alcohol metabolites.

Materials and Methods Chemicals and Reagents

Chemical and certified reference standards required for analysis werepurchased from Cerilliant (Round Rock, Tex.): acetaminophen,acetaminophen-d₄, 6-acetylmorphine, 6-acetylmorphine-d₃, alprazolam,alprazolam-d₅, amphetamine, amphetamine-d₅, benzoylecgonine,benzoylegonine-d₃, buprenorphine, buprenorphine-d₄, carisoprodol,carisoprodol-d₇, clonazepam, clonazepam-d₄, codeine, codeine-d₃,cyclobenzaprine, cyclobenzaprine-d₃, dextromethorphan,dextromethorphan-d₃, diazepam, diazepam-d₅, ethyl glucuronide (EtG),EtG-d₅, ethyl sulfate (EtS), EtS-d₅, fentanyl, fentanyl-d₅, gabapentin,gabapentin-d₁₀, hydrocodone, hydrocodone-d₃, hydromorphone,hydromorphone-d₃, ketamine, ketamine-d₅, lorazepam, lorazepam-d₄;meperidine-d₄, methylenedioxyamphetamine, methylenedioxyamphetamine-d₅,3,4-methylenedioxy-N-ethylamphetamine,3,4-methylenedioxy-N-ethylamphetamine-d₅,3,4-methylenedioxymethamphetamine, 3,4-methylenedioxymethamphetamine-d₅,meperidine, meperidine-d₄, methadone, methadone-d₃, methamphetamine,methamphetamine-d₅, methylphenidate, methylphenidate-d₉, midazolam,midazolam-d₄, morphine, morphine-d₃, naloxone, naloxone-d₅, naltrexone,naltrexone-d₃, nortriptyline, nortriptyline-d₃,o-desmethyl-cis-tramadol, o-desmethyl-cis-tramadol-d₆, oxazepam,oxazepam-d₅, oxycodone, oxycodone-d₃, oxymorphone, oxymorphone-d₃,propoxyphene, propoxyphene-d₅, phencyclidine, phencyclidine-d₅,pentazocine, quetiapine, quetiapine-d₈, tapentadol, tapentadol-d₃,temazepam, temazepam-d₅, delta-9-tetrahydrocannabinol (delta-9-THC),detal-9-THC-d₃, tramadol, tramadol-d₄, triazolam, triazolam-d₄,zaleplon, zaleplon-d₄, zolpidem, zolpidem-d₆, zopiclone andzopiclone-d₄.

Synthetic negative saliva, ELISA kits, STOP solution,3,3′,5,5′-Tetramethylbenzidine (TMB) solution, and oral fluidmulti-analyte calibrator/control sets were purchased from ImmunalysisCorporation (Pomona, Calif.). Methanol (MeOH), acetonitrile, 2-propanol,and ammonium formate were purchased from Fisher Scientific (Bridgewater,N.J.). Formic acid was purchased from Acros Organics (Bridgewater,N.J.). Types 1 and 2 water were obtained from a deionized (DI) watersystem (Millipore).

Preparation, Sampling, and Storage Conditions of Oral Fluid

Fifteen cases of suspected drug overdose investigations were performedby CHW & Pathology Associates, Inc. at Carlow University's morgue inPittsburgh, Pa. One case of suspected drug overdose investigation wasperformed in Carroll County, Carrollton, Ohio; one in Montour County,Danville, Pa.; two in Columbiana County, Lisbon, Ohio; and one in RuskCounty, Ladysmith, Wis. A Pathology Assistant carefully recorded on achain of custody form the decedent identification (ID), sex, age,weight, date, time, and location of collections. Samples of oral fluidwere collected from sublingual and submandibular sites utilizing theQuantisal Saliva Collection Device (Immunalysis Corporation). In thesubmandibular location, the collection pad was inserted into thesubmandibular gland until saturation occurred. For the sublinguallocation, the collection pad was placed under the tongue in the buccalcavity. Table 1 provides decedent demographics for twenty subjects, andoral fluid site of collection [i.e., sublingual (“SL”) or submandibular(“SM”)] by our laboratory, compared to the sample (i.e., matrix source)used by a major clinical forensic toxicology laboratory, referred toherein as “Lab X.”

TABLE 1 Decedent Demographics and Sample Matrices Decedent ID Gender AgeWeight Lab X Matrix Source Location ID Decomposed PM-1 M 55 165Peripheral Blood and SL, SM Urine PM-2 M 44 350 Peripheral Blood and SL,SM Urine PM-3 F 62 145 Peripheral Blood SL, SM PM-4 M 33 180 PeripheralBlood SL, SM PM-5 M 21 258 Hospital Blood SL, SM PM-6 M 23 135Peripheral Blood SL, SM Urine PM-7 M 67 175 Peripheral Blood SL, SM PM-8F 42 180 Peripheral Blood and SL, SM Urine PM-9 F 43 160 PeripheralBlood and SL, SM Urine PM-10 F 45 100 Peripheral Blood SL PM-11 M 53 240Peripheral Blood and SL Yes Urine PM-12 F 27 140 Peripheral Blood SL, SMPM-13 M 57 170 Bile SM Yes PM-14 M 43 185 Liver Tissue SM PM-15 F 39 120Peripheral Blood, SM Bile, and Urine PM-16 M N/A N/A Subclavical Bloodand SL Urine PM-17 M N/A N/A Blood (Unknown) SL PM-18 M N/A N/ASubclavical Blood, SL Vitreous, and Urine PM-19 M N/A N/A Autopsy BloodSL PM-20 M N/A N/A Autopsy Blood SL N/A—Not/Available

Each collection device was labeled with the appropriate sample locationID and date of collection. Each collection pad was placed into theappropriate sample location and observed for saturation of the pad forapproximately two to ten minutes. After each sample was collected, theunmodified fluid was placed into the appropriate collection device,which contained a non-azide buffer to preserve the collected oral fluid.The chain of custody form and collection devices were individuallyplaced into a dual-pocketed biohazard bag and transported to ourlaboratory to be stored in a refrigerator (2 to 8° C.) until analysiswas conducted.

After the samples were prepared for analysis, they were individuallybagged and labelled with the laboratory's sample ID and date of receipt.Samples were stored frozen in a freezer (−15 to −20° C.) for as long assix months for potential medico-legal purposes.

Preparation of Standard, Internal Standard, and Quality ControlSolutions for Analytes

All standard and internal reference standards were purchased fromCerilliant Corporation. A stock standard solution was prepared bypipetting 50 microliters (μL) of 1.0 milligram per milliliter (mg/mL) ofstandard analyte(s) and diluting them with MeOH to prepare a totalvolume of 25 mL. A stock internal standard solution was prepared bypipetting 750 μL of the 100 micrograms per milliliter (μg/mL) ofinternal standard analyte(s) and diluting the solution with MeOH toprepare a total volume of 150 mL. Three sets of Quality Control (QC)stock solutions were made to prepare three different concentrations: 10,50, and 200 nanograms per milliliter (ng/mL). The 200 ng/mL QC stocksolution was prepared by transferring 25 mL of stock standard solutioninto a graduated cylinder and diluting with synthetic negative saliva toprepare a total volume of 1 liter (L). The 10 ng/mL and 50 ng/mL QCstock solutions were prepared by serial dilutions from the 200 ng/mL QCstock solution.

Preparation of Standard, Internal Standard, and Quality ControlSolutions for Alcohol

All standard and internal reference standards were purchased fromCerilliant Corporation. A stock standard solution was prepared bypipetting 100 microliters (μL) of 1.0 milligram per milliliter (mg/mL)of standard analyte(s) and diluting them with MeOH to prepare a totalvolume of 10 mL. A stock internal standard solution was prepared bypipetting 250 μL of the 100 micrograms per milliliter (μg/mL) ofinternal standard analyte(s) and diluting the solution with MeOH toprepare a total volume of 50 mL. Three sets of Quality Control (QC)stock solutions were made to prepare three different concentrations:250, 500, and 1,000 nanograms per milliliter (ng/mL). The QC solutionwas prepared by serial dilutions utilizing SNS to prepare a total volumeof 1 mL for each concentration from the 10,000 ng/mL reference standardstock solution for the following concentrations: 2,000 ng/mL, 1,000,500, and 250 ng/mL.

Qualitative Conditions and Methodology for ELISA

The first fifteen samples were screened on a TECAN Freedom EVO 150(Tecan Group Ltd.). The following drug classes were analyzed:amphetamine, methamphetamine, opiates, propoxyphene, PCP,cocaine/benzoylecgonine, THC, benzodiazepines, tramadol, methadone,buprenorphine, fentanyl, oxycodone/oxymorphone, carisoprodol, andmeperidine. ELISA kits (Immunalysis Corporation) were utilized toqualitatively screen for the presence of analytes. The ImmunalysisDirect ELISA kit (96-well microplate) is based upon competitive bindingto the antibody of enzyme-labeled antigen and unlabeled antigen inproportion to their concentration in the reaction mixture. Samples andcalibrators were prepared by pipetting 750 μL into labeled 12×75millimeter (mm) disposable glass culture tubes (Fisher Scientific).Calibrators were prepared in duplicate (negative, low, cut-off, andhigh).

A 10 μL aliquot of diluted sample and calibrators were incubated with a100 μL of enzyme (horseradish peroxidase) into the microplate wellscoated with fixed amounts of high affinity purified polyclonal antibody.The microplates were incubated for 60 minutes at ambient temperature (20to 25° C.). Plates were washed six times with DI water utilizing amicroplate washer (TECAN). Plates were inverted and dried onto anabsorbent paper towel to remove any residual moisture. One hundred (100)μL of chromogenic substrate TMB was added. The microplates wereincubated for 30 minutes at ambient temperature and the reaction wasstopped after 30 minutes by adding 100 μL of dilute acid (STOPsolution). The plates were read with an absorbance reader (TECAN SunriseAbsorbance Reader with Magellan Software) between wavelengths of 450 to620 nanometers (nm).

Quantitative Conditions and Methods for LC-MS/MS

All samples were analyzed utilizing a 6460 Triple Quadrupole LC-MS/MScoupled with a 1290 Infinity Liquid Chromatography System (AgilentTechnologies). The column was a Poroshell 120 EC-C18 (3.0×50 mm, 2.7μm), maintained at 50° C. with a flow rate of 0.6 mL/minute. The columnfor alcohol, i.e., ethyl glucuronide (EtG) or ethyl sulfate (EtS) was aPolaris 3 C18-Ether (150×3.0 mm) maintained at 40° C. with a flow rateof 0.5 mL/minute.

The aqueous mobile phase (A) for all analytes except alcohol consistedof 5 millimolar (mM) ammonium formate and 0.1% formic acid diluted to 2L utilizing type 1 DI water; and the organic mobile phase (B) consistedof 0.1% formic acid diluted to 2 L utilizing acetonitrile. The gradientperformed is shown in Table 2.

The aqueous mobile phase (A) for alcohol consisted of 0.1% formic aciddiluted to 1,998 mL 2 L utilizing type 1 DI water. The gradientperformed is shown in Table 3.

TABLE 2 HPLC Gradient Program for Analytes Time (minutes) % A % B 0 95 51 95 5 3 70 30 6 30 70 6.5 5 95 7 99.5 0.5 8 99.5 0.5

TABLE 3 HPLC Gradient Program for Alcohol Time (minutes) % A % B 0 100 03.50 85 15 3.60 2 98 5 2 98 6 2 98

The injection volume for the samples was 2.5 μL. Mass spectrometry wasperformed utilizing a positive electrospray ionization mode for allanalytes except alcohol. The source parameters were a gas (N₂)temperature of 300° C., gas flow of 12 L/minute nebulizer pressure of 45parts per square inch (psi), sheath gas temperature of 350° C., andsheath gas flow of 12 L/minute. The ideal multiple-reaction monitoring(MRM) transitions, fragmentor voltages, collision energy voltages, andcell accelerator voltages were determined for all analytes (deuteratedand non-deuterated) by utilizing Agilent MassHunter Qualitative Analysisand Optimizer software (installed with Agilent MassHunter software). TheMRM transitions and parameters are shown in Table 4.

The injection volume for the alcohol samples was 20 μL. Massspectrometry was performed utilizing a negative electrospray ionizationmode for all analytes. The source parameters were a gas (N₂) temperatureof 300° C., gas flow of 6 L/minute nebulizer pressure of 40 parts persquare inch (psi), sheath gas temperature of 400° C., and sheath gasflow of 12 L/minute. The ideal multiple-reaction monitoring (MRM)transitions, fragmentor voltages, collision energy voltages, and cellaccelerator voltages were determined for all analytes (deuterated andnon-deuterated) by utilizing Agilent MassHunter Qualitative Analysis andOptimizer software (installed with Agilent MassHunter software). The MRMtransitions and parameters are shown in Table 4.

TABLE 4 Analytes and Alcohol with Associated Voltages for MassSpectrometry Cell MRM Fragmentor Collision Accelerator RetentionTransition Voltage Energy Voltage Time Analyte (m/z) (V) (V) (V) (min)Acetaminophen 152→110 117 15 7 1.29 152→93  117 20 7 Acetaminophen-D₄156.2→113.8 100 16 7 1.29 6-acetylmorphine 328.4→165.1 170 44 4 2.35328.4→152.1 170 80 4 6-acetylmorphine-D₃ 331.4→165.1 145 40 4 2.35Alprazolam 309.1→205.1 170 48 4 4.56 309.1→151.1 170 72 4 Alprazolam-D₅314.1→210.1 165 44 4 4.56 Amphetamine 136.2→119.1 70 4 4 2.20136.2→91.0  70 20 4 Amphetamine-D₅ 141.2→124.1 75 8 4 2.20Benzoylecgonine 290.3→168.1 135 16 4 2.71 290.3→105.0 135 32 4Benzoylecgonine-D₃ 293.3→77.1  135 68 4 2.71 Buprenorphine 468.6→115.1215 144 4 4.16 468.6→101.1 215 44 4 Buprenorphine-D₄ 473.6→473.3 215 8 44.16 Carisoprodol 261.3→176.1 75 4 4 4.52 261.3→458.1 75 4 4Carisoprodol-D₇ 268.3→183.2 80 4 4 4.52 Clonazepam 316.1→214.1 165 40 44.47 316.1→151.1 165 88 4 Clonazepam-D₄ 320.1→218.1 130 36 4 4.47Codeine 300.1→152.1 165 76 4 2.10 300.1→115.1 165 88 4 Codeine-D₃303.4→152.1 150 80 4 2.10 Cyclobenzaprine 276.1→215.1 135 48 4 4.36276.1→213.1 135 88 4 Cyclobenzaprine-D₃ 279.1→215.1 120 48 4 4.36Dextromethorphan 272.4→171.1 135 40 7 3.93 272.4→128.1 135 72 7Dextromethorphan-D₃ 275.4→128.1 105 72 7 3.93 Diazepam 285.1→193.1 16032 4 5.19 285.1→165.1 160 56 4 Diazepam-D₅ 290.1→198.1 170 32 4 5.19 EtG221.1→85   110 12 5 3.68 221.1→75   110 12 5 EtG-D₅ 226.1→75   110 12 53.56 EtS  125→96.9 90 14 5 3.31 125→80  90 34 5 EtS-D₅ 130→98  90 14 53.31 Fentanyl 337.5→188.1 130 20 4 3.86 337.5→105.1 130 48 4 Fentanyl-D₅342.5→105.1 135 48 4 3.86 Gabapentin 172.2→154.1 80 8 7 2.17 172.2→55.1 80 24 7 Gabapentin-D₁₀ 182.3→164.2 95 12 7 2.17 Hydrocodone 300.4→171.1155 40 4 2.43 300.4→128.0 155 76 4 Hydrocodone-D₃ 303.4→128.1 160 68 42.43 Hydromorphone 286.3→157.1 180 44 4 1.17 286.3→128.1 180 68 4Hydromorphone-D₃ 289.3→157.1 150 44 4 1.17 Ketamine 238.1→220.1 105 11 72.9 238.1→125.1 105 11 7 Ketamine-D₅ 242.1→129   102 15 7 2.9 Lorazepam321.1→303   136 8 7 4.52 321.1→275.1 136 21 7 Lorazepam-D₄ 325.1→77.9 115 104 7 4.52 Methylenedioxyamphetamine 180.1→162.7 75 4 7 2.42180.1→76.9  75 44 7 Methylenedioxyamphetamine-D₅ 185.1→168.1 83 5 7 2.423,4-Methylenedioxy-N- 208.1→16.1  107 9 7 2.77 ethylamphetamine208.1→105.1 107 25 7 3,4-Methylenedioxy-N- 213.2→105.1 120 30 7 2.77ethylamphetamine-D₅ 3,4-methylenedioxy- 194.3→163.1 80 12 4 2.46methamphetamine 194.3→105.1 80 24 4 3,4-methylenedioxy- 199.3→165.1 5512 4 2.46 methamphetamine-D₅ Meperidine 248.3→174.1 115 16 4 3.32248.3→103.1 115 48 4 Meperidine-D₄ 252.3→105.1 125 44 4 3.32Methamphetamine 150.2→119.1 65 8 4 2.41 150.2→91.1  65 20 4Methamphetamine-D₅ 155.2→121.2 80 8 4 2.41 Methadone 310.4→265.2 100 8 44.53 310.4→105.1 100 24 4 Methadone-D₃ 313.4→268.1 110 12 4 4.53Methylphenidate 235.2→84.1  100 16 7 3.18 235.2→56.1  116 53 7Methylphenidate-D₉ 244.3→93.1  120 16 7 3.18 Midazolam 326.1→248.6 15540 7 4.02 326.1→221.9 155 52 7 Midazolam-D₄ 330.1→249   167 40 7 4.02Morphine 286.3→165.1 155 48 4 0.82 286.3→152.1 155 68 4 Morphine-D₃289.3→152.1 170 72 4 0.82 Naloxone 328.2→310.1 120 16 7 2.13 328.2→212.1120 40 7 Naloxone-D₅ 333.4→212.1 135 40 7 2.13 Naltrexone 342.2→324.1142 20 7 2.45 342.2→55.2  142 40 7 Naltrexone-D₃ 345.2→58   105 44 72.45 Nortriptyline 264.3→202   105 60 7 4.51 264.3→91   105 24 7Nortriptyline-D₃ 267.4→91   105 24 7 4.51 o-desmethyl-cis-tramadol250.8→232.1 110 12 4 2.44 250.8→58.2  110 12 4o-desmethvl-cis-tramadol-D₆ 256.8→64.2  110 12 4 2.44 Oxazepam287.1→163.0 120 40 4 4.38 287.1→104.1 120 36 4 Oxazepam-D₅ 292.1→109.1110 40 4 4.38 Oxycodone 316.4→298.1 135 16 4 2.31 316.4→212.1 135 44 4Oxycodone-D₃ 319.4→115.1 125 104 4 2.31 Oxymorphone 302.3→198.1 130 44 40.96 302.3→128.1 130 88 4 Oxymorphone-D₃ 305.3→201.2 125 48 4 0.96Phencyclidine 244.3→159.2 80 8 4 3.74 244.3→117.1 80 32 4Phencyclidine-D₅ 249.3→164.2 80 8 4 3.74 Pentazocine* 286.4→218.1 135 164 3.57 286.4→145.1 135 36 4 Propoxyphene 340.5→266.2 80 4 4 4.49340.5→128.0 80 56 4 Propoxyphene-D₅ 345.5→271.2 80 4 4 4.49 Quetiapine384.3→220.6 145 40 7 4.03 384.3→138.8 145 96 7 Quetiapine-D₈ 392.2→258.1122 20 7 4.03 Tapentadol 222.8→108.1 130 24 4 3.13 222.8→121.0 120 15 4Tapentadol-D₃ 225.8→77.1  120 52 4 3.13 Temazepam 301.1→283.1 120 8 44.78 301.1→255.1 120 20 4 Temazepam-D₅ 306.1→260.1 120 20 4 4.78 Δ⁹-THC315→193 150 20 7 7.2 315→123 150 30 7 Δ⁹-THC-D₃ 318.4→196.3 125 20 7 7.2Tramadol 264.3→58.2  90 16 7 3.15 264.3→42.2  90 112 7 Tramadol-D₄268.4→58.2  90 16 7 3.15 Triazolam 343.1→308   190 24 7 4.65 343.1→239  190 44 7 Triazolam-D₄ 347.1→312   195 28 7 4.65 Zaleplon 306.3→64.1  13580 7 4.21 306.3→43.1  135 60 7 Zaleplon-D₄ 310.3→65.1  135 92 7 4.21Zolpidem 308.3→26.3  100 20 7 3.46 308.3→235.2 100 20 7 Zolpidem-D₆313.9→235.1 100 30 7 3.46 Zopiclone 389.8→146.1 80 16 7 3.05 389.8→112  80 64 7 Zopiclone-D₄ 393.8→246.1 80 8 7 3.05 *The internal standard forpentazocine was unavailable for purchase. Therefore, meperidine-D₄ wasutilized as the internal standard.

Sample Preparation

All samples, except alcohol samples, were filtered from the collectionpad in the Quantisal collection device. This was conducted via a bloodserum filter (16 mm×4 inches) (Porex Technologies). Five-hundred μL ofeach sample was pipetted into a LC-MS/MS vial (Fisher Scientific) with120 μL of MeOH and 30 μL of stock internal standard solution. Alcoholsamples were prepared by pipetting 540 μL of sample with 60 μL of stockinternal standard solution. Alcohol samples were prepared by pipetting540 μL of sample with 60 μL of stock internal standard solution. Afteraddition of the solutions, the vial was capped and vortexed forapproximately 10 seconds.

Method Validation

Both instruments (TECAN Freedom Evoware 150 and Agilent 6460 TripleQuadrupole LC-MS/MS coupled with a 1290 Infinity Liquid ChromatographySystem) were validated utilizing the Scientific Working Group forForensic Toxicology (SWGTOX) Standard Practices for Method Validation inForensic Toxicology. Parameters for qualitative testing (TECAN) werevalidated for limit of detection and precision. Parameters forquantitative testing (LC-MS/MS) were validated for bias, calibrationmodel, carryover, interference studies, limit of detection, limit ofquantitation, precision, dilution integrity, ionizationsuppression/enhancement, and processed sample stability.

Calibration Curve

A calibration curve was prepared to identify the unknown concentrationsof analytes in oral fluid by comparing them to a known set ofconcentrations. The concentrations for the calibration curve were 1, 5,10, 50, 100, 250, 500, and 1,000 ng/mL. The concentrations were chosenbased upon cut-offs for the analytes (see Table 4). The calibrationcurve samples were prepared as shown in Table 5.

TABLE 5 Preparation of Calibration Curve Concentrations for AnalytesOral Fluid Concentration (ng/mL) 1 5 10 50 100 250 500 1,000 Methanol367.6 337.6 300 365.6 356.2 328.0 281.2 187.6 (μL) STD (μL) — — — 9.318.8 46.9 93.8 187.5 @ 2,000 ng/mL Diluted STD  7.5  37.5   75.0 — — — —— (μL) @ 50 ng/mL

The calibration curve for analytes was prepared with 90 μL of stockinternal standard solution, 1.5 mL of synthetic negative saliva, and thespecific components listed in Table 5. After the set of concentrationswere prepared, they were vortexed for 10 seconds to ensure proper mixingof standard and internal standard solutions.

The calibration curve for alcohol is listed in Table 6. After the set ofconcentrations were prepared, they were vortexed for 10 seconds toensure proper mixing of standard and internal standard solutions.

The Oral Fluid Alcohol Reference Standard (STD) Stock Solution isprepared at a 10,000 ng/mL standard concentration. The 2,000 ng/mLstandard concentration is prepared utilizing the 10,000 ng/mL standardconcentration; the 500 ng/mL standard concentration is preparedutilizing the 2,000 ng/mL; and the 50 ng/mL standard concentration isprepared utilizing the 500 ng/mL standard concentration.

TABLE 6 Preparation of Calibration Curve Concentrations for Alcohol OralFluid Alcohol Concentration (ng/mL) 5 10 25 50 100 250 500 1000 DilutedOral Fluid Alcohol Reference 62.5 125 Standard (STD) Stock Solution (μL)@ 2000 ng/mL *Diluted Oral Fluid Alcohol Reference / / /  25  50 125 /Standard (STD) Stock Solution (μL) @ 500 ng/mL **Diluted Oral FluidAlcohol  25  50 125 / / / / / Reference Standard (STD) Stock Solution(μL) @ 50 ng/mL SNS 875 850 775 875 850 775 838.5 775 Oral Fluid AlcoholISTD (μL) 100 100 100 100 100 100 100 100 Key *Diluted Oral FluidAlcohol Reference Standard (STD) Stock Solution @ 500 ng/mL = 750 μL ofnegative oral fluid + 250 μL of diluted Oral Fluid Reference Standard(STD) Stock Solution @ 2,000 ng/mL = 1000.0 μL = 1.0 mL **Diluted OralFluid Alcohol Reference Standard (STD) Stock Solution @ 50 ng/mL = 900μL of negative oral fluid + 100 μL of diluted Oral Fluid ReferenceStandard (STD) Stock Solution @ 500 ng/mL = 1000.0 μL = 1.0 mL / = NotApplicable

Results and Discussion Sample Collection of Oral Fluid

From both the sublingual and submandibular sites where oral fluid wascollected, the collection pad absorbed approximately 1 mL of oral fluid(±10%) within 2 to 5 minutes. Oral fluid is excreted primarily by threeglands: the parotid, submaxillary and sublingual glands, as well as byother smaller glands in the head region. Oral fluid is a compositetissue comprised primarily of saliva, buccal and mucosal transudates,gingival crevicular fluid, cellular debris, bacteria, and residues ofingested products. Drug concentrations in oral fluid generally arerelated to concentrations in blood, but also may be present as residualdrug in the oral cavity. The buccal cavity contains mucous membranesthat provide a depot effect which allows absorption of higherconcentrations of certain drugs such as cocaine and amphetamines. Thislocal absorption effect is due to the higher fat solubility and ease ofpenetration through membranes with low partitioning from blood to oralfluid.

Oral Fluid Duration from Collection to Report Generation Compared to LabX

Table 7 shows the number of days from collection of samples to thereporting of the test results for our laboratory for each of the twentypost-mortem subjects compared to Lab X. The average number of days fromcollection to reporting for our laboratory ranged from 1 to 18 days,with an average of 5.7 days, compared to a range from 7 to 29 days, withan average of 16.4 days for Lab X. Thus, our laboratory was able tocollect and report out both qualitative and quantitative results usingthe methods of the present invention at least two times faster than LabX.

TABLE 7 Number of Days from Sample Collection to Report GenerationDecedent Our ID Laboratory Lab X PM-1 5 13 PM-2 5 17 PM-3 2 16 PM-4 2 18PM-5 6 9 PM-6 4 15 PM-7 4 7 PM-8 4 11 PM-9 4 18 PM-10 4 13 PM-11 4 16PM-12 4 16 PM-13 4 27 PM-14 18 29 PM-15 18 21 PM-16 1 14 PM-17 4 13PM-18 1 17 PM-19 3 14 PM-20* 16 7 *PM-20 is an outlier because a newcolumn needed to be delivered from the manufacturer.

Our validated method utilized accounts for a 1:4 dilution (oralfluid:total parts). A calibration curve and multiple sets of QC wereincluded in every analysis. Upon completion of the run, the calibratorconcentrations were analyzed using Agilent MassHunter QuantitativeAnalysis. The calibration curve was analyzed for linearity to ensure theaccuracy of the data. The linearity coefficient (R²) was greater than0.997 for each analyte of interest. The curve was analyzed for outliersand abundance of signal. QC was verified to ensure the instrumentationand technique of sample preparation was adequate.

Provided below are the test results from the twenty sets of post-mortemsamples analyzed both qualitatively and quantitatively using oral fluidas the matrix for drug testing in the fifteen post-mortem subjects,compared to the matrices collected and analyzed by Lab X, shown in Table8.

TABLE 8 Sample Matrices Analyzed by Lab X for PM 1-20 ^(a)1 PeripheralBlood ^(b)2 Urine ^(c)3 Hospital Blood ^(d)4 Blood ^(e)5 Bile ^( f)6Liver Tissue* *Concentrations measured in ng/g. “Peripheral,” “Hospital”and “Blood” are all referred to herein as “Blood.”

Post-Mortem Subject 1 Qualitative Analysis

As shown in Table 9, the post-mortem sample collection of oral fluidfrom the indicated sites in accordance with the invention detected twodrugs. Lab X detected the same two drugs and methadone collected fromblood.

TABLE 9 ELISA Results for PM-01 Presumptive Positive in Oral Fluid Lab XPresumptive Positive Benzodiazepines (SL, SM) ^(b)Benzodiazepines NotReported ^(b)Methadone Opiates (SL, SM) ^(b)Opiates

Quantitative Analysis

As shown in FIG. 1, the analysis of oral fluid yielded concentrations of6-acetylmorphine and morphine which were greater than, andconcentrations of alprazolam, codeine, and morphine which weresubstantially similar to, the concentrations of the same drugs in thissubject taken from blood reported by Lab X. In addition, we were able toquantify the concentration of methadone in oral fluid, whereas Lab Xcould not.

Post-Mortem Subject 2 Qualitative Analysis

As shown in Table 10, the post-mortem sample collection of oral fluidfrom the indicated sites in accordance with the invention detected threedrugs. Lab X's results collected from blood showed the presence of twoof the three drugs.

TABLE 10 ELISA Results for PM-2 Presumptive Positive Lab X PresumptivePositive Benzodiazepines (SL, SM) ^(b)Benzodiazepines Carisoprodol (SL,SM) Not Reported Oxycodone (SL, SM) ^(b)Oxycodone

Quantitative Analysis

As shown in FIG. 2, the analysis of oral fluid yielded concentrations ofcarisoprodol which were greater than, and concentrations of morphine andoxycodone which were substantially similar to the concentrations of thesame drugs in this subject taken from blood reported by Lab X. Inaddition, we were able to quantify the concentration of alprazolam inoral fluid, whereas Lab X could not.

Post-Mortem Subject 3 Qualitative Analysis

As shown in Table 11, the post-mortem sample collection of oral fluid inaccordance with the invention detected five drugs. Lab X's resultscollected from blood did not detect any drugs.

TABLE 11 ELISA Results for PM-3 Presumptive Positive Lab X PresumptivePositive Cannabinoids (SL, SM) Not Reported Carisoprodol (SL, SM) NotReported Cocaine/Benzoylecgonine (SL, SM) Not Reported Fentanyl (SL, SM)Not Reported Opiates (SL, SM) Not Reported

Quantitative Analysis

As shown in FIG. 3, the analysis of oral fluid yielded concentrations ofmorphine that was greater than, and concentrations of fentanyl whichwere substantially similar to concentrations of the same drugs in thissubject taken from peripheral blood reported by Lab X, except forbenzoylecgonine which was greater for Lab X. In addition, we were ableto quantify the concentration of 6-acetylmorphine and carisoprodol inoral fluid, whereas Lab X could not. Lab X also was able to quantify theconcentration of codeine in this sample.

Post-Mortem Subject 4 Qualitative Analysis

As shown in Table 12, the post-mortem sample collection of oral fluid inaccordance with the invention detected four drugs. Lab X's resultscollected from blood did not detect any drugs.

TABLE 12 ELISA Results for PM-04 Presumptive Positive Lab X PresumptivePositive Benzodiazepines (SM) Not Reported Meperidine (SM) Not ReportedOpiates (SL, SM) Not Reported Oxycodone (SL) Not Reported

Quantitative Analysis

As shown in FIG. 4, the analysis of oral fluid yielded concentrations of6-acetylmorphine, codeine, and morphine which were greater than theconcentrations of these drugs in this subject taken from blood reportedby Lab X.

Post-Mortem Subject 5 Qualitative Analysis

As shown in Table 13, the post-mortem sample collection of oral fluid inaccordance with the invention detected three drugs. Lab X's resultscollected from peripheral blood did not detect any drugs.

TABLE 13 ELISA Results for PM-5 Presumptive Positive Lab X PresumptivePositive Benzodiazepines (SL, SM) Not Reported Cannabinoids (SL, SM) NotReported Fentanyl (SL, SM) Not Reported

Quantitative Analysis

As shown in FIG. 5, the analysis of oral fluid yielded concentrations ofclonazepam and fentanyl which were substantially similar to theconcentrations of these drugs in this subject taken from blood reportedby Lab X.

Post-Mortem Subject 6 Qualitative Analysis

As shown in Table 14, the post-mortem sample collection of oral fluid inaccordance with the invention detected four drugs. Lab X's resultscollected from blood showed the presence of two of the four drugs.

TABLE 14 ELISA Results for PM-6 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Cannabinoids (SL, SM)^(b)Cannabinoids Fentanyl (SL, SM) Not Reported Opiates (SL) ^(b)Opiates

Quantitative Analysis

As shown in FIG. 6, the analysis of oral fluid yielded concentrations offentanyl which were greater than, and concentrations of morphine whichwas substantially similar to concentrations of the same drugs in thissubject taken from peripheral blood reported by Lab X. In addition, wewere able to quantify the concentration of 6-acetylmorphine in oralfluid, whereas Lab X could not.

Post-Mortem Subject 7 Qualitative Analysis

As shown in Table 15, the post-mortem sample collection of oral fluid inaccordance with the invention detected one drug. Lab X's resultscollected from blood did not detect any drugs.

TABLE 15 ELISA Results for PM-7 Presumptive Positive Lab X PresumptivePositive Fentanyl (SL, SM) Not Reported

Quantitative Analysis

As shown in FIG. 7, the analysis of oral fluid yielded a concentrationof fentanyl which was substantially similar to the concentration of thisdrug taken from blood reported by Lab X.

Post-Mortem Subject 8 Qualitative Analysis

As shown in Table 16, the post-mortem sample collection of oral fluid inaccordance with the invention detected four drugs. Lab X's resultscollected from blood detected three of these drugs, as well asoxycodone.

TABLE 16 ELISA Results for PM-8 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Cannabinoids (SL, SM)^(b)Cannabinoids Benzodiazepines (SL, SM) ^(b)Benzodiazepines Opiates(SL) ^(b)Opiates Not Reported ^(b)Oxycodone

Quantitative Analysis

As shown in FIG. 8, the analysis of oral fluid yielded concentrations of6-acetylmorphine and codeine which were not able to be quantified inthis subject taken from peripheral blood reported by Lab X. Theconcentration of oxycodone was substantially similar to concentrationsof this drug reported by Lab X. Concentrations of cyclobenzaprine andmorphine were greater from Lab X.

Post-Mortem Subject 9 Qualitative Analysis

As shown in Table 17, the post-mortem sample collection of oral fluid inaccordance with the invention detected five drugs. Lab X's resultscollected from blood showed the detection of only one of these drugs.

TABLE 17 ELISA Results for PM-9 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Benzodiazepines (SL, SM) NotReported Cocaine/Benzoylecgonine (SL) Not Reported Methadone (SL, SM)^(b)Methadone Opiates (SL) Not Reported

Quantitative Analysis

As shown in FIG. 9, the analysis of oral fluid yielded a concentrationof methadone which was greater than the concentration of this drug inthis subject taken from blood reported by Lab X. In addition, we wereable to quantify the concentrations of morphine and quetiapine in oralfluid, whereas Lab X could not.

Post-Mortem Subject 10 Qualitative Analysis

As shown in Table 18, the post-mortem sample collection of oral fluid inaccordance with the invention detected three drugs. Lab X's resultscollected from blood detected one of these drugs, as well as opiates andoxycodone.

TABLE 18 ELISA Results for PM-10 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Cannabinoids (SL, SM)^(b)Cannabinoids Methadone (SL) Not Reported Not Reported Not ReportedNot Reported ^(b)Opiates Not Reported ^(b)Oxycodone

Quantitative Analysis

As shown in FIG. 10, the analysis of oral fluid yielded concentrationsof cyclobenzaprine and oxymorphone which was greater than, and aconcentration of methadone which was substantially similar to,concentrations of these drugs in this subject taken from blood reportedby Lab X. In addition, we were able to quantify the concentration ofo-desmethyl-cis-tramadol in oral fluid, whereas Lab X could not. Lab Xwas able to quantify the concentration of oxycodone, where we did not.

Post-Mortem Subject 11 Qualitative Analysis

As shown in Table 19, the post-mortem sample collection of oral fluid inaccordance with the invention detected four drugs. Lab X's resultscollected from blood detected two of the four drugs.

TABLE 19 ELISA Results for PM-11 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Cocaine/Benzoylecgonine (SL, SM)^(b)Cocaine/Metabolites Fentanyl (SL, SM) Not Reported Opiates (SL)^(b)Opiates

Quantitative Analysis

As shown in FIG. 11, the analysis of oral fluid yielded concentrationsof benzoylecgonine and fentanyl which were substantially similar to theconcentrations of these drugs in this subject taken from blood reportedby Lab X. In addition, we were able to quantify the concentration ofmorphine in oral fluid, whereas Lab X could not.

Post-Mortem Subject 12 Qualitative Analysis

As shown in Table 20, the post-mortem sample collection of oral fluid inaccordance with the invention detected four drugs. Lab X's resultscollected from blood did not detect any drugs.

TABLE 20 ELISA Results for PM-12 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Cannabinoids (SL, SM) NotReported Fentanyl (SL, SM) Not Reported Opiates (SL) Not Reported

Quantitative Analysis

As shown in FIG. 12, the analysis of oral fluid yielded concentrationsof fentanyl, hydrocodone, and morphine which were greater than theconcentrations of these drugs in this subject taken from blood reportedby Lab X. In addition, we were able to quantify the concentration of6-acetylmorphine in oral fluid, whereas Lab X could not. Benzoylecgoninewas detected in smaller concentrations in oral fluids than was found byLab X.

Post-Mortem Subject 13 Qualitative Analysis

As shown in Table 21, the post-mortem sample collection of oral fluid inaccordance with the invention detected five drugs. Lab X's resultscollected from blood did not detect any drugs.

TABLE 21 ELISA Results for PM-13 Presumptive Positive Lab X PresumptivePositive Amphetamine (SL) Not Reported Benzodiazepines (SL) Not ReportedCocaine/Benzoylecgonine (SL) Not Reported Methamphetamine (SL) NotReported Opiates (SL) Not Reported

Quantitative Analysis

As shown in FIG. 13, the analysis of oral fluid yielded concentrationsof hydrocodone which were less than the concentration of this drug inthis subject taken from bile reported by Lab X. It should be noted thatthis decedent was highly decomposed, and thus Lab X had to determinedrug levels in the body through the invasive procedure of extractingbile from the bile duct, which is a difficult and time-consumingprocess, whereas we were able to determine the concentration ofhydrocodone rapidly in oral fluid.

Post-Mortem Subject 14 Qualitative Analysis

As shown in Table 22, the post-mortem sample collection of oral fluid inaccordance with the invention detected three drugs. Lab X's resultsusing blood did not detect any drugs.

TABLE 22 ELISA Results for PM-14 Presumptive Positive Lab X PresumptivePositive Benzodiazepines (SL) Not Reported Cocaine/Benzoylecgonine (SL)Not Reported Fentanyl (SL) Not Reported

Quantitative Analysis

As shown in FIG. 14, the analysis of oral fluid yielded a concentrationof alprazolam, whereas Lab X was not able to quantify any concentrationof this drug. Concentrations of benzoylecgonine and fentanyl were lowerin oral fluid than that found in liver tissue in this subject reportedby Lab X. No concentrations of the three drugs were found in blood byLab X. Again, it should be noted that the concentrations of the twodrugs by Lab X were found by using the invasive procedure to extractliver tissue from the decedent, a difficult and time-consuming matrix,compared to our rapid method using oral fluid.

Post-Mortem Subject 15 Qualitative Analysis

As shown in Table 23, the post-mortem sample collection of oral fluid inaccordance with the invention detected two drugs. Lab X's resultscollected from blood detected only one of these drugs.

TABLE 23 ELISA Results for PM-15 Presumptive Positive Lab X PresumptivePositive Fentanyl (SL) Not Reported Opiates (SL) ^(b)Opiates

Quantitative Analysis

As shown in FIG. 15, the analysis of oral fluid yielded concentrationsof 6-acetylmorphine, fentanyl, and morphine, in which 6-acetylmorphineand morphine were greater than, and a concentration of fentanyl whichwas substantially similar to, the concentrations of these drugs in thissubject reported by Lab X. Lab X used urine to quantitate6-acetylmorphine, and blood to quantitate morphine and fentanyl.

The following comments should be noted with respect to the foregoingresults reported for the fifteen post-mortem subjects in this study. ForPM-3, oral fluid collection from the sublingual site in the buccalcavity yielded low concentrations of codeine (4.63 ng/mL). Because itwas below the limit of quantitation, the concentration was estimated andwas not reported based upon the validation of the method for codeine.Qualitative analysis, however, showed that codeine was present. This wasobserved in decedents PM-5 with clonazepam (0.34 ng/mL), PM-10 withcyclobenzaprine and oxycodone (4.74 and 5.53 ng/mL, respectively), andPM-12 for benzoylecgonine (2.41 ng/mL). Benzoylecgonine is the mainmetabolite of the parent analyte cocaine. Because sublingual samplesfrom the buccal cavity contain large amount of lipids and fatty acids,cocaine is protected from rapid metabolism, thus yielding lowconcentrations of benzoylecgonine. Additionally, having a moreaggressive sample clean up would result in lower cut-off points and thusyield greater concentrations of drug. Additionally, PM-11 and PM-13 weredecedents that had a high degree of putrefaction (2 to 10 days,respectively). Thus, PM-13's sample location was collected only from thesublingual site in the buccal cavity. Results showed that analysescompleted by both our laboratory and Lab X resulted in similar analytes,except in PM-11 where we detected 6-acetylmorphine and morphine (6.49and 118.97 ng/mL, respectively). Lab X utilized 6-monoacetylmorphine asits analyte of choice instead of 6-acetylmorphine.

The results reported by our laboratory and Lab X show thatbenzoylecgonine, codeine, fentanyl, and morphine were common analytesreported by both laboratories. Importantly, the use of oral fluid had afurther advantage of being able to detect and report concentrations oftwo additional analytes: 6-acetylmorphine and carisoprodol, two drugsnot reported in blood from Lab X. The ability to screen and quantifythese two additional analytes, which has been shown to contribute to thecause of death, can be critical in identifying the exact cause of deathin a particular decedent. Another advantage of the use of oral fluid isin cases of post-mortem decomposition, where oral fluid is a superioralternative to blood, bile, and liver tissue, because of the process ofautolysis and the difficulty in retrieving samples for analysis fromthese matrices.

Provided below are test results from five additional post-mortem samples(PM-16, PM-17, PM-18, PM-19 and PM-20) analyzed quantitatively usingoral fluid as the matrix for drug testing in the five post-mortemsubjects, compared to blood as the matrix collected and analyzed fromthe same subjects by Lab X. In addition, seven other non-naturallyoccurring drugs underwent validation testing: zolpidem, nortriptyline,dextromethorphan, zopiclone, zaleplon, MDA and MDEA. All seven of thesedrugs were validated with respect to the use of the LC-MS/MS method toquantify non-naturally occurring drugs taken from oral fluid frompost-mortem subjects.

As shown in FIG. 16, we were able to quantify the concentration ofgabapentin in oral fluid in this subject, which concentration wasextremely high, whereas Lab X could not quantify the concentration ofthis drug taken from blood in the same subject.

As shown in FIG. 17, we were able to quantify the concentration ofnaloxone in oral fluid in this subject, whereas Lab X could not quantifythe concentration of this drug taken from blood in the same subject.

As shown in FIG. 18, we were able to quantify the concentration oftramadol in oral fluid in this subject, which concentration was veryhigh, whereas Lab X could not quantify the concentration of this drugtaken from blood in the same subject.

As shown in FIG. 19, we were able to quantify the concentration ofacetaminophen in oral fluid in this subject, which concentration wasextremely high, whereas Lab X could not quantify the concentration ofthis drug taken from blood in the same subject.

As shown in FIG. 20, we were able to quantify the concentrations of EtSand EtG in oral fluid in this subject, whereas Lab X could not quantifythe concentrations of these drugs and/or drug metabolites taken fromblood in the same subject.

Provided below are test results from six live dogs (SF10085, SF10086,SF10087, SF10088, SF10092, SF10048) and from two post-mortem dogs(SF10093, SF10049) analyzed quantitatively using oral fluid as thematrix for drug testing. The non-naturally occurring drugs whichunderwent validation testing with respect to the use of the LC-MS/MSmethod to quantify non-naturally occurring drugs taken from oral fluidfrom post-mortem subjects were ketamine, methamphetamine, diazepam,acetaminophen, tramadol and gabapentin.

As shown in FIG. 21, ketamine was detected and quantified in oral fluidat a concentration of 598.30 ng/mL in a live dog (SF10085).

As shown in FIG. 22, ketamine was detected and quantified in oral fluidat a concentration of 592.10 ng/mL in a live dog (SF10086).

As shown in FIG. 23, ketamine was detected and quantified in oral fluidat a concentration of 1914.90 ng/mL in a live dog (SF10087).

As shown in FIG. 24, ketamine was detected and quantified in oral fluidat a concentration of 426.20 ng/mL in a live dog (SF10088).

As shown in FIG. 25, ketamine was detected and quantified in oral fluidat a concentration of 844.20 ng/mL in a live dog (SF10092).

As shown in FIG. 26, ketamine was detected and quantified in oral fluidat a concentration of 229.50 ng/mL in a post-mortem dog (SF10093).

As shown in FIG. 27, methamphetamine was detected and quantified in oralfluid at a concentration of 16.20 ng/mL in a live dog (SF10048).

As shown in FIG. 28, in a post-mortem dog (SF10049), diazepam wasdetected and quantified in oral fluid at a concentration of 4.00 ng/mL,ketamine was detected and quantified in oral fluid at a concentration of408.30 ng/mL, acetaminophen was detected and quantified in oral fluid ata concentration of 12.20 ng/mL, tramadol was detected and quantified inoral fluid at a concentration of 49.70 ng/mL, and gabapentin wasdetected and quantified in oral fluid at a concentration of 205.00ng/mL.

It should be noted that the present invention is not limited to thespecific analytes, drugs or drug metabolites disclosed herein, butincludes, without limitation, drugs from the following non-limiting drugclasses: opioids, benzodiazepines, antidepressants, antihistamines,antipsychotics, anticonvulsants, muscle relaxants, barbiturates,stimulants, hypnotics and illicit drugs.

Further, sample collection of oral fluid from post-mortem subjects andfrom live and post-mortem animals in accordance with the presentinvention demonstrates the surprising sensitivity of this collectionmethod in detecting drugs of interest. Our methodology using oral fluidwas shown to be about three times more effective in detecting drugs ofinterest qualitatively compared to the use of conventional matrices suchas blood, urine, bile, and liver tissue, as shown by the resultsobtained by Lab X. Thus, the present invention using oral fluidcollection provides a surprisingly sensitive method for qualitativeanalysis of samples from post-mortem subjects and from live andpost-mortem animals. The results of the qualitative drug screening inaccordance with the invention then can be further confirmed andquantified utilizing analytical instrumentation.

After qualitative analysis of the post-mortem samples, oral samples wereanalyzed from the post-mortem subjects and from live and post-mortemanimals utilizing LC-MS/MS instrumentation to confirm and quantitateconcentrations of the analytes initially detected. This is in contrastto quantitative analyses performed by laboratories using conventionalmatrices for drug analysis, where various instrumentations are requiredto quantitate, as well as screen, drug samples collected from thesematrices. Comparison of the instrumentations used in the presentinvention using oral fluid, and that used by Lab X using conventionalmatrices, for both qualitative and quantitative analyses, is shown inTable 24.

It should be noted that the present invention is not limited to theinstrumentation disclosed herein (i.e., ELISA and LC-MS/MS), butincludes any suitable instrumentation capable of screening for andquantifying analytes in post-mortem subjects.

TABLE 24 Instrumentation Used by Our Laboratory Compared to Lab XDecedent ID Our Instrumentation Lab X Instrumentation PM-1 ELISA,LC-MS/MS LC-MS/MS, GC/MS, GC, EIA, ELISA, LC/TOF-MS PM-2 ELISA, LC-MS/MSLC/TOF-MS, HPLC, GC/MS, LC-MS/MS, EIA, ELISA, GC PM-3 ELISA, LC-MS/MSLC-MS/MS, GC-GC-GC/MS, GC/MS, LC/TOF-MS, GC PM-4 ELISA, LC-MS/MSLC-MS/MS, ELISA, GC, LC/TOF-MS PM-5 ELISA, LC-MS/MS LC-MS/MS,GC-GC-GC/MS, HPLC, ELISA, GC, LC/TOF- MS PM-6 ELISA, LC-MS/MSGC-GC-GC/MS, LC-MS/MS, EIA, ELISA, GC, LC/TOF-MS PM-7 ELISA, LC-MS/MSLC-MS/MS, ELISA, GC, LC/TOF-MS PM-8 ELISA, LC-MS/MS LC-MS/MS, GC-GC-GC/MS, GC, EIA, ELISA, LC/TOF-MS PM-9 ELISA, LC-MS/MS LC-MS/MS, GC/MS,HPLC, EIA, ELISA, GC, LC/TOF-MS PM-10 ELISA, LC-MS/MS GC-GC-GC/MS,GC/MS, LC- MS/MS, EIA, ELISA, GC, LC/TOF-MS PM-11 ELISA, LC-MS/MSGC-GC-GC/MS, GC/MS, LC- MS/MS, EIA, ELISA, LC/TOF- MS, GC PM-12 ELISA,LC-MS/MS GC-GC-GC/MS, GC/MS, LC- MS/MS, ELISA, GC, LC/TOF- MS PM-13ELISA, LC-MS/MS LC-MS/MS, GC-GC-GC/MS, GC, ELISA, C PM-14 ELISA,LC-MS/MS GC/MS, HPLC, LC-MS/MS, GC, C, ELISA PM-15 ELISA, LC-MS/MSLC-MS/MS, GC, ELISA, LC/TOF-MS, EIA PM-16 LC-MS/MS N/A PM-17 LC-MS/MSEIA, GC, LCMS PM-18 LC-MS/MS GC/MS PM-19 LC-MS/MS N/A PM-20 LC-MS/MS GCCorrelation and t-Test Statistics

Correlation statistics were calculated to analyze the data gathered byour laboratory and Lab X, and are shown in Table 25.*

TABLE 25 Correlation and Comparison Statistics for Sublingual and Lab XSamples (ng/mL) Degrees Sample of Correlation Decedent Lab X Lab XFreedom Coefficient P- ID Mean Variance Mean Variance (df) (r) valuePM-1 343.03 314,741.43 180.00 12,0486.50 7 0.393 0.598 PM-2 478.381,188,492.02 872.50 4,095,437.50 8 0.278 0.686 PM-3 182.75 61,274.74293.83 410,472.17 6 0.533 0.706 PM-4 17,624.49 253,428,848.19 73.6311,978.80 2 0.441 0.196 PM-5 1.74 3.94 6.80 9.68 2 0.342 0.192 PM-642.43 425.01 20.67 321.33 4 0.880 0.240 PM-7 N/A N/A N/A N/A N/A 0.095N/A PM-8 55.91 3,582.17 81.80 11,941.20 6 0.848 0.659 PM-9 1,179.842,349,418.07 305.00 186,050.00 1 0.260 0.579 PM-10 10.01 358.18 20.53579.23 9 0.649 0.422 PM-11 197.29 179,842.61 224.20 239,778.20 8 0.2640.928 PM-12 35.69 2,335.83 89.67 38,561.87 6 0.517 0.538 PM-13 N/A N/AN/A N/A N/A 0.228 N/A PM-14 34.89 2,051.28 183.33 46,233.33 34.89 0.4000.363 PM-15 78.47 944.18 37.67 1,196.33 4 0.906 0.201 PM-16 N/A N/A N/AN/A N/A N/A N/A PM-17 N/A N/A N/A N/A N/A N/A N/A PM-18 N/A N/A N/A N/AN/A N/A N/A PM-19 N/A N/A N/A N/A N/A N/A N/A PM-20 N/A N/A N/A N/A N/AN/A N/A N/A (Not Applicable) *PM-16 to PM-20 not subjected tostatistical analysis because of inability of Lab X to quantify anyconcentration of drugs or drug metabolites in these subjects.

The mean and variance calculated for our results were for the sublinguallocation only. The mean and variance calculated for Lab X results werefor all matrices reported. All fifteen post-mortem samples showed apositive correlation between the data collected by our laboratory andLab X. This indicates a direct relationship between the two sets of data(i.e., if one set increases, the other set increases). As shown in Table10, statistics (t-test) were utilized for data comparison. An importantfinding was that concentration levels were not equivalent between ourlaboratory and Lab X, as our methodology resulted in higherconcentrations (i.e., was more sensitive) in most cases.

P-values were calculated by our laboratory to determine the probabilityof rejection of the test hypothesis that oral fluid and blood aredirectly related to each other. Because the p-values for all decedentsamples were greater than 0.05 (p>0.05), the testing hypothesis couldnot be rejected, which definitively showed that oral fluid and bloodsampling are directly and proportionately related to one another,confirming that oral fluid is a viable alternative to conventionalmethods of sampling. Further, the results from our laboratory using oralfluid samples, when compared the results from Lab X, confirms that notonly is oral fluid a viable alternative, but indeed a superioralternative, being faster, sensitive, more consistent, and less invasivethan conventional methods.

Conclusion

This study demonstrated that oral fluid collected from various sites isnot only an equivalent, but a superior, alternative to traditionalmethods of fluid and tissue collections for post-mortem drug analysissubjects and from live and post-mortem animals, due to the ease ofcollection, simpler use of instrumentation, safety concerns, andrapidity and quality of results obtained. Because conventionalpost-mortem analyses are performed mainly with blood as the matrix, drugconcentrations often may vary in unpredictable ways based upon thecollection site of the blood, time of sampling, and the phenomenon ofredistribution. The sampling compartment is assumed to relate to theconcentration at the site of action. After death, however, compartmentsare usually altered as the integrity of the compartmental barriers islost. This, in turn, may alter the concentration of one or more drugsoriginally contained in the intact compartments, leading to erroneousdrug testing results (i.e., either the non-finding of a particular drugin a particular matrix), or the finding of lowered or skewedconcentration levels. In contrast, the use of oral fluid as the primaryanalytical matrix for post-mortem drug testing eliminates these errorfactors, as oral fluid is seen to be surprisingly preserved after deathin all the sites in which oral fluid may be collected. In other words,the sites reported herein in which oral fluid was collected appear toserve as intact reservoirs for oral fluid, so that any drugs containedin the oral fluid are able to be analyzed with enhanced sensitivity,consistency and accuracy compared to conventional methodologies. Inaddition, the use of oral fluid to detect and quantify non-naturallyoccurring drugs in live animals is much less stressful to animalscompared to the invasive method of using blood as the matrix.

The ease of collection eliminates the necessity to collect additionalfluids and tissue, and thus accelerates the autopsy process, whichallows for cases to be closed substantially faster, as, for example,examination of larvae and entomological samples associated withputrefied cases may cause increased expense and time to determine causeof death. This study demonstrates that oral fluid can be collectedrapidly and easily by procedures that do not interfere with medicalexamination and that are less time consuming relative to the collectionprocedures associated with blood, urine, bile, and other fluids.Further, the use of oral fluid samples requires only one screening andanalytical technique. This is in contrast to conventional methods whichrequire the use of numerous matrices, as well as numerous screening andanalytical techniques. Thus, because the methodology of the presentinvention requires the use of only one matrix (i.e., oral fluid) and twoinstrumentalities to obtain sensitive and consistent results, theresults can be reported significantly faster than conventional methodsusing conventional matrices and requiring the use of a number ofinstruments.

EXAMPLES

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

The following two examples report forensic toxicology results on a62-year old female decedent weighing 145 pounds suspected of dying froma drug overdose, using the methodology of the present invention.

Example 1—Oral Fluid Collection from Sublingual Area

A cellulose collection pad from a Quantisal Saliva Collection Device wasplaced into the sublingual area adjacent to the second bicuspid andfirst molar in the buccal cavity of the decedent for approximately threeto five minutes. The collection pad was removed and observed forsaturation of the pad with oral fluid. The collection pad was placedinto the collection device, containing a non-azide buffer. The coroner'sID for the subject, date, and sample location was written onto thecollection device and placed into a dual-pocketed zippered biohazardbag. The collection device was placed into the zippered pocket of thebag and the chain of custody was folded and placed into the opposingpocket. All samples were placed into a United Parcel Services Laboratory(UPS) Pak and then shipped to the laboratory for analyses. Upon receiptin the laboratory, the sample was inspected for viability, sufficientquantity of oral fluid, and paperwork. The samples were prepared under anegative pressure hood and filtered using a blood serum filter (16 mm×4inches).

Qualitative results were derived using a Direct ELISA kit (96-wellmicroplate), which is based upon the competitive binding to antibody ofenzyme-labeled antigen and unlabeled antigen in proportion to theirconcentration in the reaction mixture. Samples and calibrators wereprepared by pipetting 750 μL into labeled 12×75 mm disposable glassculture tubes (Fisher Scientific). Calibrators were prepared induplicate (negative, low, cut-off, and high). Qualitative results werecompleted in approximately three hours and compiled for reporting to thecoroner's office.

Quantitative results were completed using a 6460 LC-MS/MS. The resultsthen were reviewed twice by a laboratory scientist and reported to thecoroner's office within approximately two to five days upon receipt ofthe sample. The following drugs and their concentrations were detectedin the sublingual region: 6-acetylmorphine, 109.69 ng/mL;benzoylecgonine, 669.59 ng/mL; carisoprodol, 49.20 ng/mL; codeine, 5.64ng/mL; fentanyl, 70.62 ng/mL; and morphine, 197.42 ng/mL. The resultswere compared to forensic toxicology results detected in blood of thesame decedent by Lab X.

The results obtained with oral fluid collected from the sublingualregion of the subject were found to be substantially similar to orsuperior with respect to the number of drugs detected qualitatively andconcentrations of the drugs detected quantitatively compared to resultsobtained from blood collected from the same decedent by Lab X.

Example 2—Oral Fluid Collection from Submandibular Gland

During the initial autopsy preparation procedure, an incision dissectionwas made to expose the submandibular gland of the decedent. An incisionthen was made in the gland and a cellulose collection pad from aQuantisal Saliva Collection Device was inserted into the gland forapproximately five to ten minutes. The collection pad was removed andobserved for saturation of the pad with oral fluid. The collection padwas placed into the collection device containing a non-azide buffer. Thecoroner's ID for the subject, date, and sample location was written ontothe collection device and placed into a dual-pocketed zippered biohazardbag. The collection device was placed into the zippered pocket of thebag and the chain of custody was folded and placed into the opposingpocket. All samples were placed into a UPS Laboratory Pak and thenshipped to the laboratory for analyses. Upon receipt into thelaboratory, the sample was observed for viability, sufficient oral fluidquantity, and paperwork. The samples were prepared under a negativepressure hood and filtered using a blood serum filter (16 mm×4 inches).

Qualitative results were derived using a Direct ELISA kit (96-wellmicroplate), which is based upon the competitive binding to antibody ofenzyme-labeled antigen and unlabeled antigen in proportion to theirconcentration in the reaction mixture. Samples and calibrators wereprepared by pipetting 750 μL into labeled 12×75 mm disposable glassculture tubes (Fisher Scientific). Calibrators were prepared induplicate (negative, low, cut-off, and high). Qualitative results werecompleted in approximately 3 hours and compiled for reporting to thecoroner's office.

Quantitative results were completed using a 6460 LC-MS/MS. The resultsthen were reviewed twice by a laboratory scientist and reported to thecoroner's office within approximately 24 to 72 hours upon receipt of thesample. The following drugs and their concentrations were detected inthe submandibular gland: 6-acetylmorphine, 0.0 ng/mL; benzoylecgonine,269.02 ng/mL; carisoprodol, 54.38 ng/mL; codeine, 0.0 ng/mL; fentanyl,11.41 ng/mL; and morphine, 12.34 ng/mL. The results were compared toforensic toxicology results detected in blood of the same decedent byLab X.

The results obtained with oral fluid collected from the submandibulargland of the subject were found to be substantially similar to orsuperior with respect to the number of drugs detected qualitatively andconcentrations of the drugs detected quantitatively compared to resultsobtained from blood collected from the same decedent by Lab X.

Example 3—Oral Fluid Collection from Sublingual Area of a Live Dog

Sedation of the dog with 30 mg/kg pentobarbital administeredintravenously preceded the collection procedure. A cellulose collectionpad from a Quantisal Saliva Collection Device was placed into thesublingual area adjacent to the second bicuspid and first molar in thebuccal cavity of the dog for three minutes. The collection pad wasremoved and observed for saturation of the pad with oral fluid. Thecollection pad was placed into the collection device, containing anon-azide buffer. The ID for the subject, date, and sample location waswritten onto the collection device and placed into a dual-pocketedzippered biohazard bag. The collection device was placed into thezippered pocket of the bag and the chain of custody was folded andplaced into the opposing pocket. All samples were placed into a UnitedParcel Services Laboratory (UPS) Pak and then shipped to the laboratoryfor analyses. Upon receipt in the laboratory, the sample was inspectedfor viability, sufficient quantity of oral fluid, and paperwork. Thesamples were prepared under a negative pressure hood and filtered usinga blood serum filter (16 mm×4 inches).

Quantitative results were completed using a 6460 LC-MS/MS. The resultsthen were reviewed twice by a laboratory scientist and reported back toour laboratory within four hours. The following drug was detected andquantified in the sublingual region: ketamine, 598.30 mL.

Example 4—Oral Fluid Collection from Sublingual Area of a Post-MortemDog

A cellulose collection pad from a Quantisal Saliva Collection Device wasplaced into the sublingual area adjacent to the second bicuspid andfirst molar in the buccal cavity of the dog for three minutes. Thecollection pad was removed and observed for saturation of the pad withoral fluid. The collection pad was placed into the collection device,containing a non-azide buffer. The ID for the subject, date, and samplelocation was written onto the collection device and placed into adual-pocketed zippered biohazard bag. The collection device was placedinto the zippered pocket of the bag and the chain of custody was foldedand placed into the opposing pocket. All samples were placed into aUnited Parcel Services Laboratory (UPS) Pak and then shipped to thelaboratory for analyses. Upon receipt in the laboratory, the sample wasinspected for viability, sufficient quantity of oral fluid, andpaperwork. The samples were prepared under a negative pressure hood andfiltered using a blood serum filter (16 mm×4 inches).

Quantitative results were completed using a 6460 LC-MS/MS. The resultsthen were reviewed twice by a laboratory scientist and reported back toour laboratory within four hours. The following drugs were detected andquantified in the sublingual region: diazepam, 4.00 ng/mL; ketamine,408.30 ng/mL; acetaminophen, 12.20 ng/mL; tramadol, 49.70 ng/mL; andgabapentin, 205.00 ng/mL.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A rapid, sensitive method for the detection andquantification of non-naturally occurring drugs for forensic drugtesting in a live or a post-mortem animal using oral fluid from thepost-mortem animal, comprising: collecting a sample of oral fluid from alive or a post-mortem animal; analyzing the oral fluid samplequantitatively using a Liquid Chromatography-Mass Spectrometry/MassSpectrometry (LC-MS/MS) method to determine concentration of the one ormore non-naturally occurring drugs in the live or the post-mortemanimal; and identifying the one or more non-naturally occurring drugs inthe live or the post-mortem animal, wherein detection and quantificationin oral fluid is more sensitive and faster than quantification of thenon-naturally occurring drugs in urine, blood, bile and liver tissue inthe same live or post-mortem animal using the LC-MS/MS method, saidnon-naturally occurring drugs classified in drug classes consisting ofnon-steroidal anti-inflammatory drugs (NSAIDs), alcohol, alcoholmetabolites, barbiturates, benzodiazepines, synthetic cannabinoids,cathinones, general anesthetics, muscle relaxants, neuroleptics,opiates, semi-synthetic opioids, opioid antagonists/analgesics,stimulants, hypnotics, antitussives, antidepressants, cannabinoids,antipsychotics, anticonvulsants, antihistamines and illicit drugs,wherein quantitative results are obtained in as soon as three hours. 2.The method of claim 1, wherein the drug classes and the non-naturallyoccurring drugs classified in the drug classes are analgesics comprisingnon-steroidal anti-inflammatory drugs (NSAIDs) comprising acetaminophen;benzodiazepines comprising diazepam; general anesthetics comprisingketamine; neuroleptics comprising gabapentin; semi-synthetic opioidscomprising tramadol; and illicit drugs comprising methamphetamine. 3.The method of claim 1, wherein the sample of oral fluid is collectedfrom a buccal cavity of the live or the post-mortem animal.
 4. Themethod of claim 3, wherein the sample of oral fluid is collected from asublingual region of the buccal cavity.
 5. The method of claim 1,wherein the sample of oral fluid is collected from a submandibulargland.
 6. The method of claim 1, wherein the sample of oral fluid iscollected with a collection pad in about one minute to about tenminutes.
 7. The method of claim 1, wherein the sample of oral fluid thatis collected is about 1 millimeter (mL).
 8. The method of claim 1,wherein the live or the post-mortem animal is selected from the groupconsisting of dogs, cats, horses and farm animals.
 9. The method ofclaim 8, wherein the live or the post-mortem animal is a dog.